Background and Significance Expression of HER2 receptors in breast cancers is correlated with poor prognosis and their expression may be different in distant metastases as compared to the primary tumor. This project will provide means to assess global expression of HER2 in breast cancers (including metastases) and to deliver therapeutic agents specifically to HER2-positve cells. As the targeting agent we propose to use Affibody molecules obtained from our CRADA partner in Sweden (http://www.affibody.com). These very stable and highly soluble alpha-helical proteins are relatively small (8.3 kDa) and can be readily expressed in bacterial systems or produced by peptide synthesis. The His6-Zher2:324 binds to HER2 receptors with high affinity (22 pM) and is available with cystein at the carboxy-terminal to facilitate conjugation. For imaging purposes, these molecules with be labeled with radionuclides. For therapy, the His6-Zher2:324 will be conjugated with thermo-sensitive liposomes that, labeled with beacons for in vivo imaging and loaded with therapeutic agents (e.g. toxins, radiosensitizers or kinase inhibitors), will allow local drug release defined by real-time monitoring of their distribution. In spite of repeated attempts to use Lipososmes for site-directed delivery of hydrophilic and hydrophobic drugs for cancer therapy, their application is limited due to poor understanding of lipososmes interactions with the cells and the sub-optimal biodistribution profile of various formulations. We will circumvent these problems using multifunctional liposomes with targeting, imaging and optimal drug release capabilities. Our strategy, involving assessment of target presence and distribution in an individual patient followed by optimized, target-specific drug delivery, may significantly improve efficacy of breast cancer treatment while reducing side effects. Experimental procedures Labeling with imaging agents The Affibody molecules are conjugated using maleimide chemistry with either AlexaFluor or 18F for, respectively, optical and PET imaging. Thermosensitive liposomes Liposomes with optimal lipid composition, sensitive to temperatures just above 37oC. The thermal destabilization of liposomes in a temperature range of 37oC-45oC will be determined by monitoring calcein leakage by spectrofluorometry methods. The next step will include optimization of techniques to conjugate HER2-specific Affibody molecules to the liposome surface via the maleimide group and their labeling with optical, PET, and MRI imaging agents. As the liposome size is an important determinant of their biodistribution, we will characterize the size of the resulting conjugates using the resources at Nanotechnology Characterization Laboratory. The liposomes will be loaded with therapeutic agents according to published methods. We will consult clinical oncologists at CCR regarding the best drugs available for treatment of breast cancers. In vitro and in vivo characterization Using binding, proliferation and clonogenic survival assays, as well as molecular biology methods we will thoroughly characterize in vitro the binding properties of the conjugates and their effects on the target cells. We will also investigate the impact of their binding on receptor expression and binding of therapeutic antibodies (Herceptin). Biodistribution of the conjugates will be studied using nude mice bearing xenografts of HER2-positive tumors. To test the in vivo imaging capacity of radioconjugates, we will monitor, by optical or PET imaging, the expected downregulation of HER2 in tumor xenografts following treatment with Herceptin or DMAG. The estimation of expression level obtained from imaging data will be verified by ex-vivo analysis of tumor tissue by immunohistochemistry and Western blots. Initial in vivo studies of our drug delivery system will be carried out using liposomes loaded with a combination of hydrophilic and hydrophobic fluorescent markers allowing to map and track in vivo tissue/organ compartmentalization of liposomes and to monitor drug leakage and/or release at 37oC-45oC. Noninvasive methods based on focused ultrasound techniques will be optimized to disintegrate liposomes. MRI, and micro-PET may be used to optimize the imaging capacity of the conjugates by those modalities. Experimental therapy of HER2-positive tumor-bearing animals will be carried out to assess the improvement of the efficacy of therapeutic agents delivered by tumor targeting, heat sensitive liposomes as compared with current application methods. For Exploratory IND Studies a single mammalian species can be used to establish a margin of safety. Accomplishments 1. Methods for labeling of HER2-specific Affibody molecules with fluorine-18 have been developed and published in the Journal of Fluorine Chemistry. 2. 18F-Affibody conjugates have been characterized in vitro and in vivo as reported in the European Journal of Nuclear Medicine and Molecular Imaging. 3. Different types of HER2-specific Affibody molecules (monomer, dimer, and albumin binding domain containing) have been labeled with AlexaFluor dyes and characterized in vitro and in vivo as reported in Clinical Cancer Research. 4. Thermosensitive liposomes have been developed and conjugated with HER2-specific Affibody molecules. A manuscript describing the design and physico-chemical characteristics of the resulting nanoparticles has been accepted for publication in the Journal of Liposome Research 5. Biodistribution studies of 18F-labeled Affibody in primates have been performed. 6. Subcutaneous models of tumors with different levels of HER2 expression have been established and used to assess the dependence of signal observed in imaging studies on the HER2 expression. 7. PET imaging was successfully used to quantify changes in HER2 expression following treatment of tumor bearing mice with 17-DMAG and the resulting manuscripot have been submitted for publication in the Journal of Nuclear Medicine.